Single-primer nucleic acid amplification methods

• US 7,374,885 B2
• Filed: 08/26/2005
• Issued: 05/20/2008
• Est. Priority Date: 08/27/2004
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Brentano, Steven T., Livezey, Kristin W., Becker, Michael M., Lam, Wai-Chung, Kolk, Daniel P., Schroder, Astrid R. W.
• Primary Examiner(s): Kim; Young J.
• Assistant Examiner(s): Thomas; David C.
• Application Number:
  US11/213,519
• Publication Number:
  US 20060046265A1
• Time in Patent Office: 998 Days
• Field of Search: None
• US Class Current: 435/5
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2537/163 blocking probe

Abstract

The present invention is directed to novel methods of synthesizing multiple copies of a target nucleic acid sequence which are autocatalytic (i.e., able to cycle automatically without the need to modify reaction conditions such as temperature, pH, or ionic strength and using the product of one cycle in the next one). In particular, the present invention discloses a method of nucleic acid amplification which is robust and efficient, while reducing the appearance of side-products. The method uses only one primer, the “priming oligonucleotide,” a promoter oligonucleotide modified to prevent polymerase extension from its 3′-terminus and, optionally, a means for terminating a primer extension reaction, to amplify RNA or DNA molecules in vitro, while reducing or substantially eliminating the formation of side-products. The method of the present invention minimizes or substantially eliminates the emergence of side-products, thus providing a high level of specificity. Furthermore, the appearance of side-products can complicate the analysis of the amplification reaction by various molecular detection techniques. The present invention minimizes or substantially eliminates this problem, thus providing an enhanced level of sensitivity.

Claims

1. A method of synthesizing multiple copies of a target sequence, said method comprising the steps of:

(A) treating a target nucleic acid comprising an RNA target sequence with;

(1) a priming oligonucleotide which hybridizes to the 3′

-end of said target sequence such that a primer extension reaction can be initiated therefrom, wherein at least one of the following conditions is satisfied;

(a) said priming oligonucleotide does not comprise RNA; and

(b) said priming oligonucleotide has a cap hybridized to a 3′

-end thereof prior to hybridizing to said target sequence, said cap comprising a base region which is complementary to at least 3 nucleotides at the 3′

-end of said priming oligonucleotide, wherein the 5′

-terminal base of said cap is complementary to the 3′

-terminal base of said priming oligonucleotide, and wherein said cap is modified to prevent the initiation of DNA synthesis therefrom; and

(2) a binding molecule which binds to said target nucleic acid adjacent to or near the 5′

-end of said target sequence;

(B) extending said priming oligonucleotide in a primer extension reaction with a DNA polymerase to give a DNA primer extension product complementary to said target sequence, said DNA primer extension product having a 3′

-end which is determined by said binding molecule and which is complementary to the 5′

-end of said target sequence;

(C) separating said DNA primer extension product from said target sequence using an enzyme which selectively degrades said target sequence;

(D) treating said DNA primer extension product with a promoter oligonucleotide comprising first and second regions, said first region hybridizing to a 3′

-region of said DNA primer extension product to form a promoter oligonucleotide;

DNA primer extension product hybrid, and said second region being a promoter for an RNA polymerase and situated 5′

to said first region, wherein any oligonucleotide provided in said method which comprises a promoter for an RNA polymerase is modified to prevent the initiation of DNA synthesis therefrom;

(E) extending the 3′

-end of said DNA primer extension product in said promoter oligonucleotide;

DNA primer extension product hybrid to add a sequence complementary to said second region of said promoter oligonucleotide; and

(F) transcribing from said promoter oligonucleotide;

DNA primer extension product hybrid multiple RNA products complementary to said DNA primer extension product using an RNA polymerase which recognizes said promoter and initiates transcription therefrom, wherein the base sequences of said RNA products are substantially identical to the base sequence of said target sequence.

2. The method of claim 1, wherein the activity of said DNA polymerase in said method is substantially limited to the formation of said DNA primer extension product.

3. The method of claim 1, wherein said priming oligonucleotide has said cap hybridized to a 3′

-end thereof prior to hybridizing to said target sequence.

4. The method of claim 3, wherein said cap is complementary to no more than 8 nucleotides at the 3′

-end of said priming oligonucleotide.

5. The method of claim 3, wherein said cap prevents non-specific hybridization between said priming oligonucleotide and said promoter oligonucleotide when said cap is hybridized to said priming oligonucleotide.

6. The method of claim 3, wherein said cap is a capping oligonucleotide modified to include a blocking moiety situated at its 3′

-terminus.

7. The method of claim 3, wherein the 3′

-end of said cap is covalently attached to the 5′

-end of said priming oligonucleotide, and wherein said cap hybridizes to the 3′

-end of said priming oligonucleotide by forming a loop.

8. The method of claim 7, wherein said cap is joined to said priming oligonucleotide via a linker region.

9. The method of claim 8, wherein said linker region comprises at least 5 nucleotides.

10. The method of claim 8, wherein said linker region comprises at least 5 abasic nucleotides.

11. The method of claim 1, wherein said priming oligonucleotide does not comprise RNA.

12. The method of claim 11, wherein said priming oligonucleotide consists of deoxynucleotides and/or analogs thereof.

13. The method of claim 12, wherein said priming oligonucleotide consists of deoxynucleotides.

14. The method of claim 1, wherein said priming oligonucleotide does not include a non-hybridizing base region situated 5′

to said base region which hybridizes to said target sequence.

15. The method of claim 14, wherein a 5′

-region of said priming oligonucleotide includes one or more modifications for increasing the binding affinity of said priming oligonucleotide for said target sequence, and wherein said modifications do not prevent said priming oligonucleotides from being extended in a primer extension reaction.

16. The method of claim 15, wherein said modifications are spaced at least 15 bases from the 3′

-terminus of said priming oligonucleotide.

17. The method of claim 15, wherein said modifications are selected from the group consisting of 2′

-O-methylribonucleotides and LNAs.

18. The method of claim 1, wherein said priming oligonucleotide is extended using a reverse transcriptase having an RNAse H activity.

19. The method of claim 18, wherein said reverse transcriptase is derived from Maloney murine leukemia virus.

20. The method of claim 1, wherein said enzyme has an RNAse H activity, and wherein said enzyme is other than a reverse transcriptase.

21. The method of claim 1, wherein said binding molecule comprises an oligonucleotide having a blocking moiety situated at its 3′

-terminus to prevent the initiation of DNA synthesis therefrom.

22. The method of claim 21, wherein the 5′

-end of said oligonucleotide of said binding molecule overlaps the 5′

-end of said first region of said promoter oligonucleotide, and wherein the 5′

-end of said first region has a sufficient number of mismatches with the 5′

-end of said oligonucleotide of said binding molecule to prevent said promoter oligonucleotide from hybridizing to said binding molecule.

23. The method of claim 21, wherein said binding molecule includes one or more modifications for increasing the binding affinity of said binding molecule for said target nucleic acid.

24. The method of claim 23, wherein said binding molecule includes one or more 2′

-O-methylribonucleotides or LNAs.

25. The method of claim 21, wherein said binding molecule is a terminating oligonucleotide.

26. The method of claim 25, wherein the 5′

end of said terminating oligonucleotide is complementary to at least two nucleotides at the 5′

end of said first region of said promoter oligonucleotide.

27. The method of claim 25, wherein the 5′

end of said terminating oligonucleotide is complementary to at least three but no more than ten nucleotides at the 5′

end of said first region of said promoter oligonucleotide.

28. The method of claim 21, wherein said binding molecule is a modifying oligonucleotide.

29. The method of claim 28, wherein said modifying oligonucleotide is a digestion oligonucleotide.

30. The method of claim 1 further comprising treating said DNA primer extension product with an extender oligonucleotide in step (d), said extender oligonucleotide hybridizing to a region of said DNA primer extension product 3′

to said promoter oligonucleotide of said promoter oligonucleotide;

DNA primer extension product hybrid, such that an extender oligonucleotide;

DNA primer extension product hybrid is formed.

31. The method of claim 30, wherein said extender oligonucleotide further comprises a blocking moiety situated at its 3′

terminus to prevent the initiation of DNA synthesis therefrom.

32. The method of claim 30, wherein said extender oligonucleotide hybridizes to said DNA primer extension product such that the 5′

-terminal base of said extender oligonucleotide is spaced within three nucleotides of the 3′

-terminal base of said promoter oligonucleotide relative to said DNA primer extension product.

33. The method of claim 30, wherein said extender oligonucleotide hybridizes to said primer extension product adjacent said promoter oligonucleotide in step (D).

34. The method of claim 1, wherein said promoter oligonucleotide further comprises an insertion sequence positioned between or adjacent to said first and second regions, and wherein the presence of said insertion sequence in said promoter oligonucleotide enhances the rate at which said RNA products are formed.

35. The method of claim 34, wherein said insertion sequence comprises the nucleotide base sequence of SEQ ID NO:

1 or SEQ ID NO;

2.

36. The method of claim 1, wherein said promoter oligonucleotide is modified to include a blocking moiety situated at its 3′

-terminus.

37. The method of claim 36, wherein said blocking moiety of said promoter oligonucleotide comprises a substituent selected from the group consisting of:

a modified nucleotide, a nucleotide or a nucleotide sequence having a 3′

-to-5′

orientation, a 3′

alkyl group, a 3′

2′

-dideoxynucleotide, a 3′

cordycepin, a 3′

alkane-diol residue, a 3′

non-nucleotide motiey, a nucleotide sequence non-complementary to the target sequence, a nucleic acid binding protein, and mixtures thereof.

38. The method of claim 36, wherein said blocking moiety does not comprise a 3′

2′

-dideoxynucleotide or a 3′

-terminus having a free hydroxyl group.

39. The method of claim 36, wherein said blocking moiety comprises a nucleotide or a nucleotide sequence having a 3′

-to-5′

orientation or a 3′

-non-nucleotide moiety.

40. The method of claim 1 further comprising the step of determining the presence or amount of said multiple copies of said target sequence.

41. The method of claim 40, wherein the presence or amount of said multiple copies of said target sequence is determined with an oligonucleotide probe having a detectable label.

42. The method of claim 41, wherein the presence or amount of said multiple copies of said target sequence is determined after step (F).

43. The method of claim 41, wherein the presence or amount of said multiple copies of said target sequence is determined during step (F).

44. The method of claim 43, wherein said probe is a self-hybridizing probe and includes a pair of interacting labels.

45. The method of claim 1 further comprising the step of determining the presence or amount of said DNA primer extension product.

46. The method of claim 1, wherein said method is carried out at a substantially constant temperature.

47. A method of synthesizing multiple copies of a target sequence, said method comprising the steps of:

(A) treating a target nucleic acid comprising an RNA target sequence with a priming oligonucleotide which hybridizes to the 3′

-end of said target sequence, such that a primer extension reaction can be initiated therefrom;

(B) extending said priming oligonucleotide in a primer extension reaction with a DNA polymerase to give a first DNA primer extension product having an undefined 3 ′

-end and comprising a base region complementary to said target sequence;

(C) separating said first DNA primer extension product from said target nucleic acid using an enzyme which selectively degrades that portion of said target nucleic acid which is complementary to said first DNA primer extension product;

(D) treating said first DNA primer extension product with a promoter oligonucleotide comprising first and second regions, said first region hybridizing to a 3′

-region of said first DNA primer extension product to form a promoter oligonucleotide;

first DNA primer extension product hybrid, and said second region being a promoter for an RNA polymerase and situated 5′

to said first region, wherein any oligonucleotide provided in said method which comprises a promoter for an RNA polymerase is modified to prevent the initiation of DNA synthesis therefrom, and wherein said first DNA primer extension product is not extended to form a double-stranded promoter comprising said promoter; and

(E) transcribing from said promoter oligonucleotide;

first DNA primer extension product hybrid multiple first RNA products complementary to at least a portion of said first DNA primer extension product using an RNA polymerase which recognizes said promoter and initiates transcription therefrom, wherein the base sequences of said first RNA products are substantially identical to the base sequence of said target sequence.

48. The method of claim 47, wherein the activity of said DNA polymerase in said method is substantially limited to the formation of primer extension products comprising said priming oligonucleotide.

49. The method of claim 47 further comprising the steps of:

(F) treating one of said first RNA products produced in step (E) with said priming oligonucleotide to form a priming oligonucleotide;

first RNA product hybrid such that a primer extension reaction can be initiated from said priming oligonucleotide;

(G) extending said priming oligonucleotide of step (F) in a primer extension reaction with said DNA polymerase to give a second DNA primer extension product complementary to said first RNA product, said second DNA primer extension product having a 3′

-end which is complementary to the 5′

-end of said first RNA product;

(H) separating said second DNA primer extension product from said first RNA product using an enzyme which selectively degrades said first RNA product;

(I) treating said second DNA primer extension product with said promoter oligonucleotide to form a promoter oligonucleotide;

second DNA primer extension product hybrid;

(J) extending the 3′

-end of said second DNA primer extension product in said promoter oligonucleotide;

second DNA primer extension product hybrid of step (I) to add a sequence complementary to said second region of said promoter oligonucleotide; and

(K) transcribing from said promoter oligonucleotide;

second DNA primer extension product hybrid of step (J) multiple second RNA products complementary to said second DNA primer extension product using said RNA polymerase, wherein the base sequences of said second RNA products are substantially identical to the base sequence of said RNA target sequence.

50. The method of claim 47, wherein said priming oligonucleotide has said cap hybridized to a 3′

-end thereof prior to hybridizing to said target sequence, said cap comprising a base region which is complementary to at least 3 nucleotides at the 3′

-end of said priming oligonucleotide, wherein the 5′

-terminal base of said cap is complementary to the 3′

-terminal base of said priming oligonucleotide, and wherein said cap is modified to prevent the initiation of DNA synthesis therefrom.

51. The method of claim 50, wherein said cap is complementary to no more than 8 nucleotides at the 3′

-end of said priming oligonucleotide.

52. The method of claim 50, wherein said cap prevents non-specific hybridization between said priming oligonucleotide and said promoter oligonucleotide when said cap is hybridized to said priming oligonucleotide.

53. The method of claim 50, wherein said cap is a capping oligonucleotide modified to include a blocking moiety situated at its 3′

-terminus.

54. The method of claim 50, wherein the 3′

-end of said cap is covalently attached to the 5′

-end of said priming oligonucleotide, and wherein said cap hybridizes to the 3′

-end of said priming oligonucleotide by forming a loop.

55. The method of claim 54, wherein said cap is joined to said priming oligonucleotide via a linker region.

56. The method of claim 55, wherein said linker region comprises at least 5 nucleotides.

57. The method of claim 55, wherein said linker region comprises at least 5 abasic nucleotides.

58. The method of claim 47, wherein said priming oligonucleotide does not comprise RNA.

59. The method of claim 58, wherein said priming oligonucleotide consists of deoxynucleotides and/or analogs thereof.

60. The method of claim 59, wherein said priming oligonucleotide consists of deoxynucleotides.

61. The method of claim 47, wherein said priming oligonucleotide does not include a non-hybridizing base region situated 5′

to said base region which hybridizes to said target sequence.

62. The method of claim 61, wherein a 5′

-region of said priming oligonucleotide includes one or more modifications for increasing the binding affinity of said priming oligonucleotide for said target sequence, and wherein said modifications do not prevent said priming oligonucleotide from being extended in a primer extension reaction.

63. The method of claim 62, wherein said modifications are spaced at least 15 bases from the 3′

-terminus of said priming oligonucleotide.

64. The method of claim 62, wherein said modifications are selected from the group consisting of 2′

-O-methylribonucleotides and LNAs.

65. The method of claim 47, wherein said priming oligonucleotide is extended using a reverse transcriptase having an RNAse H activity.

66. The method of claim 65, wherein said reverse transcriptase is derived from Maloney murine leukemia virus.

67. The method of claim 47, wherein said enzyme has an RNAse H activity, and wherein said enzyme is other than a reverse transcriptase.

68. The method of claim 47 further comprising the step of treating said first DNA primer extension product with an extender oligonucleotide in step (D), said extender oligonucleotide hybridizing to a region of said first DNA primer extension product 3′

to said promoter oligonucleotide of said promoter oligonucleotide;

first DNA primer extension product hybrid, such that an extender oligonucleotide;

first DNA primer extension product hybrid is formed.

69. The method of claim 68, wherein said extender oligonucleotide further comprises a blocking moiety situated at its 3′

-terminus to prevent the initiation of DNA synthesis therefrom.

70. The method of claim 68, wherein said extender oligonucleotide hybridizes to said first DNA primer extension product such that the 5′

-terminal base of said extender oligonucleotide is spaced within three nucleotides of the 3′

-terminal base of said promoter oligonucleotide relative to said first DNA primer extension product.

71. The method of claim 68, wherein said extender oligonucleotide hybridizes to said first DNA primer extension product adjacent said promoter oligonucleotide in step (D).

72. The method of claim 47, wherein said promoter oligonucleotide further comprises an insertion sequence positioned between or adjacent to said first and second regions, and wherein the presence of said insertion sequence in said promoter oligonucleotide enhances the rate at which said first RNA products are formed.

73. The method of claim 72, wherein said insertion sequence comprises the nucleotide base sequence of SEQ ID NO:

1 or SEQ ID NO;

2.

74. The method of claim 47, wherein said promoter oligonucleotide is modified to include a blocking moiety situated at its 3′

-terminus.

75. The method of claim 74, wherein said blocking moiety of said promoter oligonucleotide comprises a substituent selected from the group consisting of:

a modified nucleotide, a nucleotide or a nucleotide sequence having a 3′

-to-5′

orientation, a 3′

alkyl group, a 3′

2′

-dideoxynucleotide, a 3′

cordycepin, a 3′

alkane-diol residue, a 3′

non-nucleotide moiety, a nucleotide sequence non-complementary to the target sequence, a nucleic acid binding protein, and mixtures thereof.

76. The method of claim 75, wherein said blocking moiety does not comprise a 3′

2′

-dideoxynucleotide or a 3′

-terminus having a free hydroxyl group.

77. The method of claim 75, wherein said blocking moiety comprises a nucleotide or a nucleotide sequence having a 3′

-to-5′

orientation or a 3′

-non-nucleotide moiety.

78. The method of claim 47 further comprising the step of determining the presence or amount of said multiple copies of said target sequence.

79. The method of claim 78, wherein the presence or amount of said multiple copies of said target sequence is determined with an oligonucleotide probe having a detectable label.

80. The method of claim 79, wherein the presence or amount of said multiple copies of said target sequence is determined after step (E).

81. The method of claim 79, wherein the presence or amount of said multiple copies of said target sequence is determined during step (E).

82. The method of claim 81, wherein said probe is a self-hybridizing probe and includes a pair of interacting labels.

83. The method of claim 49 further comprising the step of determining the presence or amount of said first DNA primer extension product, said second DNA primer extension product, or mixtures thereof.

84. The method of claim 47, wherein said method is carried out at a substantially constant temperature.

85. A method of synthesizing multiple copies of a target sequence, said method comprising the steps of:

(A) treating a target nucleic acid comprising a DNA target sequence with a promoter oligonucleotide comprising first and second regions, said first region hybridizing to the 3′

-end of said target sequence to form a promoter oligonucleotide;

target nucleic acid hybrid, and said second region being a promoter for an RNA polymerase and situated 5′

to said first region, wherein any oligonucleotide provided in said method which comprises a promoter for an RNA polymerase is modified to prevent the initiation of DNA synthesis therefrom, and wherein said target nucleic acid is not extended to form a double-stranded promoter comprising said promoter;

(B) transcribing from said promoter oligonucleotide;

target nucleic acid hybrid multiple first RNA products comprising a base region complementary to said target sequence using an RNA polymerase which recognizes said promoter and initiates transcription therefrom;

(C) treating one of said first RNA products with a priming oligonucleotide which hybridizes to a 3′

-region of said first RNA product, such that a primer extension reaction can be initiated therefrom;

(D) extending said priming oligonucleotide in a primer extension reaction with a DNA polymerase to give a DNA primer extension product complementary to at least a portion of said first RNA product, said DNA primer extension product having a 3′

-end which is complementary to the 5′

-end of said first RNA product;

(E) separating said DNA primer extension product from said first RNA product using an enzyme which selectively degrades said first RNA product;

(F) treating said DNA primer extension product with said promoter oligonucleotide to form a promoter oligonucleotide;

DNA primer extension product hybrid; and

(G) transcribing from said promoter oligonucleotide;

DNA primer extension product hybrid multiple second RNA products complementary to said DNA primer extension product using said RNA polymerase, wherein the base sequences of said second RNA products are substantially complementary to the base sequence of said target sequence.

86. The method of claim 85, wherein the activity of said DNA polymerase in said method is substantially limited to the formation of said DNA primer extension product.

87. The method of claim 85, wherein said priming oligonucleotide has said cap hybridized to a 3′

-end thereof prior to hybridizing to said first RNA product, said cap comprising a base region which is complementary to at least 3 nucleotides at the 3′

-end of said priming oligonucleotide, wherein the 5′

-terminal base of said cap is complementary to the 3′

-terminal base of said priming oligonucleotide, and wherein said cap is modified to prevent the initiation of DNA synthesis therefrom.

88. The method of claim 87, wherein said cap is complementary to no more than 8 nucleotides at the 3′

-end of said priming oligonucleotide.

89. The method of claim 87, wherein said cap prevents non-specific hybridization between said priming oligonucleotide and said promoter oligonucleotide when said cap is hybridized to said priming oligonucleotide.

90. The method of claim 87, wherein said cap is a capping oligonucleotide modified to include a blocking moiety situated at its 3′

-terminus.

91. The method of claim 87, wherein the 3′

-end of said cap is covalently attached to the 5′

-end of said priming oligonucleotide, and wherein said cap hybridizes to the 3′

-end of said priming oligonucleotide by forming a loop.

92. The method of claim 91, wherein said cap is joined to said priming oligonucleotide via a linker region.

93. The method of claim 92, wherein said linker region comprises at least 5 nucleotides.

94. The method of claim 93, wherein said linker region comprises at least 5 abasic nucleotides.

95. The method of claim 85, wherein said priming oligonucleotide does not comprise RNA.

96. The method of claim 95, wherein said priming oligonucleotide consists of deoxynucleotides and/or analogs thereof.

97. The method of claim 96, wherein said priming oligonucleotide consists of deoxynucleotides.

98. The method of claim 85, wherein said priming oligonucleotide does not include a non-hybridizing base region situated 5′

to said base region which hybridizes to said target sequence.

99. The method of claim 98, wherein a 5′

-region of said priming oligonucleotide includes one or more modifications for increasing the binding affinity of said priming oligonucleotide for said first RNA product, and wherein said modifications do not prevent said priming oligonucleotide from being extended in a primer extension reaction.

100. The method of claim 99, wherein said modifications are spaced at least 15 bases from the 3′

-terminus of said priming oligonucleotide.

101. The method of claim 99, wherein said modifications are selected from the group consisting of 2′

-O-methylribonucleotides and LNAs.

102. The method of claim 85, wherein said priming oligonucleotide is extended using a reverse transcriptase having an RNAse H activity.

103. The method of claim 102, wherein said reverse transcriptase is derived from Maloney murine leukemia virus.

104. The method of claim 85, wherein said enzyme has an RNAse H activity, and wherein said enzyme is other than a reverse transcriptase.

105. The method of claim 85 further comprising the steps of:

treating said target nucleic acid with an extender oligonucleotide in step (A), said extender oligonucleotide hybridizing to a region of said target nucleic acid 3′

to said promoter oligonucleotide of said promoter oligonucleotide;

target nucleic acid hybrid such that an extender oligonucleotide;

target nucleic acid hybrid is formed; and

treating said DNA primer extension product with an extender oligonucleotide in step (F), said extender oligonucleotide hybridizing to a region of said DNA primer extension product 3′

to said promoter oligonucleotide of said promoter oligonucleotide;

DNAprimer extension product hybrid such that an extender oligonucleotide;

DNA primer extension product hybrid is formed.

106. The method of claim 105, wherein said extender oligonucleotide of each of steps (A) and (F) further comprises a blocking moiety situated at its 3′

-terminus to prevent the initiation of DNA synthesis therefrom.

107. The method of claim 105, wherein:

said extender oligonucleotide of step (A) hybridizes to said target nucleic acid such that the 5′

-terminal base of said extender oligonucleotide is spaced within three nucleotides of the 3′

-terminal base of said promoter oligonucleotide relative to said target nucleic acid; and

said extender oligonucleotide of step (F) hybridizes to said DNA primer extension product such that the 5′

-terminal base of said extender oligonucleotide is spaced within three nucleotides of the 3 ′

-terminal base of said promoter oligonucleotide relative to said DNA primer extension product.

108. The method of claim 105, wherein:

said extender oligonucleotide of step (A) hybridizes to said target nucleic acid adjacent said promoter oligonucleotide; and

said extender oligonucleotide of step (F) hybridizes to said DNA primer extension product adjacent said promoter oligonucleotide.

109. The method of claim 85, wherein said promoter oligonucleotide further comprises an insertion sequence positioned between or adjacent to said first and second regions, and wherein the presence of said insertion sequence in said promoter oligonucleotide enhances the rate at which said RNA products are formed.

110. The method of claim 109, wherein said insertion sequence comprises the nucleotide base sequence of SEQ ID NO:

1 or SEQ ID NO;

2.

111. The method of claim 85, wherein said promoter oligonucleotide is modified to include a blocking moiety situated at its 3′

-terminus.

112. The method of claim 111, wherein said blocking moiety of said promoter oligonucleotide comprises a substituent selected from the group consisting of:

a modified nucleotide, a nucleotide or a nucleotide sequence having a 3′

-to-5′

orientation, a 3′

alkyl group, a 3′

2′

-dideoxynucleotide, a 3′

cordycepin, a 3′

alkane-diol residue, a 3′

non-nucleotide moiety, a nucleotide sequence non-complementary to the target sequence, a nucleic acid binding protein, and mixtures thereof.

113. The method of claim 111, wherein said blocking moiety does not comprise a 3′

2′

-dideoxynucleotide or a 3′

-terminus having a free hydroxyl group.

114. The method of claim 111, wherein said blocking moiety comprises a nucleotide or a nucleotide sequence having a 3′

-to-5′

orientation or a 3′

-non-nucleotide moiety.

115. The method of claim 85 further comprising the step of determining the presence or amount of said multiple copies of said target sequence.

116. The method of claim 115, wherein the presence or amount of said multiple copies of said target sequence is determined with an oligonucleotide probe having a detectable label.

117. The method of claim 116, wherein the presence or amount of said multiple copies of said target sequence is determined after step (G).

118. The method of claim 116, wherein the presence or amount of said multiple copies of said target sequence is determined during steps (B) and (G).

119. The method of claim 118, wherein said probe has a stem-loop structure and includes a pair of interacting labels.

120. The method of claim 85 further comprising the step of determining the presence or amount of said DNA primer extension product.

121. The method of claim 85, wherein said method is carried out at a substantially constant temperature.